For a systematic analysis of tissue protease activity using MALDI MSI the undecapeptide substance P (RPKPQQFFGLM; [M+H]+ = m/z 1347.7354) was selected
as tracer substrate. Porcine tissues (brain, kidney, liver, pancreas, spleen and muscle) were tested with if for unspecific whole-tissue protease activity.
Homogeneity assessment of porcine tissue
H&E stains or representative tissue slices reveal the inhomogeneity of brain tissue (Figure 9). Inhomogeneity can be problematic for the spatial analysis of the experimental setup. The homogeneity of all other tissues is a prerequisite for the topically applied inhibitor spots in the following experimental setup.
Figure 9: H&E stain for porcine tissue All porcine tissue were H&E stained according to standard staining
protocol. The slides were scanned with high resolution using the Aperio CS2 (Leica Biosystems) scanner. Morphology of the tissue showed inhomogeneity for brain tissue, represented by white and grey matter. The other tissue blocks showed good homogeneity.
Specificity analysis for the tracer peptide and related ions on porcine tissue It is another prerequisite that specificity of substance P signals is given for all tissues. All porcine tissues were measured using MALDI-TOF MS instrument (UltrafleXtreme, Bruker Daltonics) without any application of substance P. The tissue mean spectra did not show any signal that potentially could interfere with substance P or substance P– related signals (Figure 10).
Figure 10: Specificity of m/z values of substance P and its related peptides on porcine tissue Shown are
MALDI MSI time course visualising tissue protease activity of porcine tissues By spray coating, porcine liver tissue with substance P and further incubation at 37 °C and with 95% humidity (in SunDigest Incubation chamber) for 30 min a consistent decrease of the substance P signal and an increase in substance P-related peptide signals were observed (Figure 11), compared to a control without incubation. The experiment was designed by myself and conducted by Kevin Reinle in the context of his masterthesisf. The detected increasing masses could be assigned to the C-terminal
cleavage products of substance P (Table 6). This first result led to the hypothesis, that the digestion of the tracer substance P by endogenous proteases, preserved in frozen tissue, could be visualised.
Table 6: Substance P related peptides C-terminally cleaved
Substance P 1347.7 m/z RPKPQ QFFGL M SubP(1-10) 1217.7 m/z RPKPQ QFFGL SubP(1-9) 1104.6 m/z RPKPQ QFFG SubP(1-8) 1047.6 m/z RPKPQ QFF SubP(1-7) 900.5 m/z RPKPQ QF SubP(1-6) 753.4 m/z RPKPQ Q SubP(1-5) 625.4 m/z RPKPQ
To verify this hypothesis, an experimental setup for all six porcine tissues was developed, where time-dependency of the digestion could be observed with MALDI MSI. Further, a protease inhibitor mix (PIM) was pipetted (4 x 1 µL in 1x working solution, concentrations of single inhibitors according to Table 3) on the tissue, to indicate a protease-related change of signal intensity (Figure 4). Incubation was performed in the SunDigest incubation chamber.
f Masterthesis Kevin Reinle, Visualising endogeneous protease activity by MALDI Mass Spectrometry
Figure 11: Comparison spectra digest/no digest show increased fragments of substance P In an initial
experiment substance P was sprayed on pancreas tissue – one prior to incubation at 37 °C and 95% humidity, for 30 min (digest), the other after this incubation (no digest). The fold-change plot revealed that the digested tissue showed decreased substance P signals (in red) compared to no-digest tissue. The set of substance P signals consist of the protonated form, adducts with sodium and potassium, as well as their respective isotopologues. Further, m/z values increased in the digested tissue (green) that might correspond to cleaved products of substance P.
The first observation was that on muscle tissue there was no difference between substance P signal distribution on no digest- and digest-slide. This means that no reduction of the substance P – signal, thus no digestion could be observed (Figure 12). Further, the spotted PIM did not have an impact on the substance P signal distribution in both conditions.
There was a difference in the absolute intensity scale when measuring the time points on different days. To normalise this variability, the no digest control was always measured with the digest slide. This makes a comparison of the different time-points possible. Nevertheless, when the no digest control visually did not show a homogeneous distribution of the substance P signal, the data set was excluded for further analysis.
Figure 12: MALDI MSI of substance P and resulting peptides on porcine muscle Muscle tissue cryosections
of 10 µm were mounted on two ITO-slides. Protease inhibitor mix was pipetted onto each tissue (4x 1 µL). The “digest” and “no-digest” slides were spray-coated with substance P before and after incubation (15, 30, 60, 120, 360 min), respectively. After coating with DHB, MALDI-TOF MSI was performed at 200 µm spatial resolution. For muscle tissue the ion intensity distribution of substance P (m/z 1347.7) on the “digest” slide did not change over time. Scale bar is 2 mm.
Figure 13: MALDI MSI of substance P and resulting peptides on spleen Tissue was sliced in 10 µm sections
and placed on two ITO-slides (“digest”-slide and “no-digest”-slide). Protease-Inhibitor-Mix was pipetted onto each tissue (4x 1 µL). The “digest”-slide was spray-coated with substance P before and the “no-digest”-slide after incubation at 37 °C, 95 % humidity (incubation times: 15, 30, 60, 120, 360 minutes). After coating with MALDI matrix, the acquisition was performed on a MALDI-TOF MS (UltrafleXtreme, Bruker Daltonics) with 200 µm spatial resolution. MALDI-TOF MS images of the “no-digest”-slide showed a homogeneous distribution of substance P. This description holds true for the following figures. On the “digest” slide, the spots with inhibitor showed higher substance P signals than the surrounding tissue without inhibitor. Where low substance P signal intensity was detected, peptides corresponding to m/z values 1104.6, 900.5 and 753.4 are visible. In the case of
The porcine tissues spleen, liver, pancreas and kidney (Figure 13 to Figure 16) showed a time-dependent reduction and a homogeneous distribution of the substance P signal on the no digest controls. The spots, where PIM was pipetted, were clearly visible and displayed a higher substance P signal compared to the non-inhibited surrounding tissue. In detail, when looking at the MALDI MSI of substance P signal distribution on spleen after 15 min incubation, very low signal intensity (represented in blue colour) could be seen in the non-inhibited regions. In contrast in the PIM spots the intensity was preserved. After 120 min of incubation the substance P signal was decreased in the PIM spots also, until it was gone after 360 min incubation. The absolute depletion of substance P signal holds true for all tissue, apart from muscle.
Figure 14: MALDI MSI of substance P and resulting peptides on liver See description of Figure 13. On the
“digest” slide, the spots with inhibitor showed higher substance P signals than the surrounding tissue without inhibitor. Where low substance P signal intensity was detected, peptides corresponding to m/z values 1104.6, 900.5 and 753.4 are visible. In case of m/z 900.5 the inhibition retained the decrease of the signal at 120 min incubation. Scale bar is 2 mm.
Figure 15: MALDI MSI of substance P and resulting peptides on pancreas See description of Figure 13. On
the “digest” slide spots with inhibitor showed higher substance P signals than the surrounding tissue without inhibitor. Where low substance P signal intensity was detected, peptides corresponding to m/z values 1104.6, 900.5 and 753.4 are visible. Pancreas seemingly shows a poor reaction on the spotted inhibitor. Scale bar is 2 mm.
Figure 16: MALDI MSI of substance P and resulting peptides on kidney See description of Figure 13. On
the “digest” slide spots with inhibitor showed higher substance P signals than the surrounding tissue without inhibitor. Where low substance P signal intensity was detected the m/z values 1104.6, 900.5 and 753.4 are visible. The inhibition retains the decrease of the signal m/z 900.5 at 120 min incubation. Scale bar is 2 mm
As previously mentioned homogeneous distribution of substance P on the no-digest control is the prerequisite for a proper statistical analysis. According to the H&E stain of brain tissue (Figure 9), the brain sample consists of white and grey matter. This inhomogeneity resulted in inhomogeneous distribution of the signal, most likely due to different ionisation and suppression effects in the two tissue types123. This generated
very unreliable and unstable results for this dataset.
Figure 17: MALDI MSI of substance P and resulting peptides on brain See description of Figure 13. MALDI
MSI of the “no-digest”-slide showed inhomogeneous distribution of substance P, due to morphological differences in brain tissue.
The MALDI MSI of the time-curve experiment did not only reveal the protease- dependent decrease of substance P signal (as PIM retains the decrease), but also the time-dependent occurrence of substance P-related peptides. The most intense were SubP(1-5) m/z 753.4, SubP(1-7) m/z 900.5 and SubP(1-9) m/z 1104.6. With m/z 1104.6 and 753.4 being dominantly produced in the non-inhibited tissue, m/z 900.5 is produced in the PIM spots. This could be optimally observed on spleen (Figure 13). Here, after 360 min incubation no difference between inhibited and non-inhibited regions was detectable anymore. The time-dependent production of peptides suggests that substance P is truly digested rather than sequestered into the tissue.
Digest efficiency calculation in the time course experiments
An absolute analysis of the MALDI MS Images is difficult. All time-points and tissue relative digest efficiencies (DE%) were calculated according to equation (2). Mean spectra were extracted from the four PIM spots (+ inhibitor) and from tissue without PIM (- inhibitor). The substance P intensity for no digest and digest slide were also extracted. The time-curve experiments were performed a second time under slightly varied conditions: The incubation was done in a standard cell-culture oven at 37 °C (Figure 18) instead of the SunDigest incubation chamberg (Figure 19)
Figure 18: Time-curves of digest efficiency of porcine tissues in a cell culture incubator All tissue showed
a very fast increase of DE% when no inhibitor was used (continuous line). The Protease Inhibitor Mix (PIM) slowed down the digestion process (broken line). Pancreas did not react on the PIM and showed a very fast digestion process. Muscle tissue showed no decrease in substance p signal and was thus used as negative control.
g Experiments in SunDigest chamber were designed by myself and conducted by Kevin Reinle in the framework of his Masterthesis: Visualising endogeneous protease activity by MALDI Mass Spectrometry Imaging, 2017
Figure 19: Time-courses of digest efficiency of porcine tissues in the incubation chamber All tissue
showed a very fast increase of DE% when no inhibitor was used (continuous line). The Protease Inhibitor Mix (PIM) retarded the digestion process (broken line). Pancreas did not react on the PIM and showed a very fast digestion process. Muscle tissue showed no decrease in substance p signal and was thus used as negative control.
The calculated DE% in the cell culture incubator showed already 100% digestion of the substance P signal after 15 min incubation, lasting on this level in all tissues (instead of muscle) (Figure 18). In the SunDigest chamber a nearly 100% digestion for all tissues (instead of muscle) was reached after 60 min incubation time (Figure 19). It seemed that the conditions in the SunDigest chamber favoured a slow progress of the proteolytic activity. Both experiments showed decreased DE% when PIM was applied to the tissue. Further, brain showed a high standard deviation according to its tissue heterogeneity (data not shown). Porcine pancreas showed no effect on PIM and high variance (< 1 h), regardless of the incubation condition. This was possibly due to the complexity of protease constitution in this highly active digestive tissue124. In spleen
and liver tissue PIM-induced retardation of substance P degradation was highly significant. Muscle tissue showed no reduction of the substance P signal in both conditions, so the calculation of DE% did not show an increase.
Generation of substance P cleavage products in time course experiment
It was observed that substance P related peptides occurred transiently during the time course. Ion intensities for m/z 1104.6, m/z 900.5 and m/z 753.4 were extracted in
MALDI MSI (Figure 12 to Figure 17) the m/z value 900.5 was predominantly produced in inhibited regions (blue line, Figure 20). The other two fragments were produced predominantly without inhibitor. This finding suggested that different sets of proteases are responsible:
1. Without PIM: m/z 1347.7 1104.6 753.4 X 2. With PIM: m/z 1347.7 900.5 X
In brain, the standard deviations were very high, so no difference could be observed.
Figure 20: Transient presence of substance P cleavage products is modulated with PIM TIC-normalised ion
intensities were extracted from mean spectra of porcine tissue of the areas without (orange line) and with (blue line) protease inhibitor mix (PIM) (S/N>5). The intensities of putative substance P cleavage products m/z 1104.6, 900.5 and 753.4 that are generated transiently are plotted versus the incubation time. Note that m/z 900.5 was recorded with higher intensity in PIM containing regions.
The effect of PIM indicated that the reduction of substance P signal was not due to uncontrolled effects like sequestering or adsorption of substance P into the tissue, but rather was related to real protease-driven digestion.
4.1.4 Inhibitor-concentration dependency of proteolytic activity in porcine